Espacenet — Bibiiographic data
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`6/28/2016 12:48 AM
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`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`24 August 2006 (24.08.2006)
`
` (10) International Publication Number
`
`WO 2006/089203 A2
`
`(51) International Patent Classification:
`
`Not classified
`
`(21) International Application Number:
`PCT/US2006/005792
`
`(22) International Filing Date:
`16 February 2006 (16.02.2006)
`
`I—00030
`Louis, David [IT/IT]; Piazzale Clementi #5,
`Genazzano, Rome (IT). UMANSKY, Samuil, R. [US/US];
`3 Orchid Court, Princeton, New Jersey 08540 (US).
`Agent: ELRIFI, Ivor, R.; Mintz, Levin, Cohn, Ferris,
`Glovsky and Popeo PC, One Financial Center, Boston,
`Massachusetts 02111 (US).
`
`(74)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`RM2005 A000068
`11/137,935
`
`17 February 2005 (17.02.2005)
`25 May 2005 (25.05.2005)
`
`IT
`US
`
`(63)
`
`Related by continuation (CON) or continuation-impart
`(CIP) to earlier applications:
`US
`Filed on
`US
`Filed on
`
`RM2005A000068 (CIP)
`17 February 2005 (17.02.2005)
`11/137,935 (CIP)
`25 May 2005 (25.05.2005)
`
`(71) Applicant (for all designated States except US): ISTI-
`TUTO NAZIONALE PER LE MALATTIE INFET-
`TIVE IRCCS LAZZARO SPALLANZANI [IT/IT]; Via
`Portuense, 292, 1—00149Rome (IT).
`
`(72) Inventors; and
`HOVSEP,
`only):
`(for US
`(75) Inventors/Applicants
`Melkonyan [RU/US]; 546 Ewing Street, Princeton,
`New Jersey 08540 (US). CANNAS, Angela [IT/IT]; Via
`Genne’s Frongia N. 29,
`I—09031 Arbus (IT). TOMEI,
`
`(81)
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KN, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
`LY, MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI,
`NO, NZ, OM, PG, PII, PL, PT, RO, RU, SC, SD, SE, SG,
`SK, SL, SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US
`(patent), UZ, VC, VN, YU, ZA, ZM, ZW.
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, lVIZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (Alyl, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, 'l‘G).
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`(54) Title: COMPOSITIONS AND METHODS FOR DETECTING VIRAL SPECIFIC NUCLEIC ACIDS IN URINE
`
`[Continued on next page]
`
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`79 bp
`
`(57) Abstract: The present invention
`relates
`to methods
`for
`diagnosis
`or monitoring 01' viral
`inl'ection by
`detecting the presence of transrenal
`Viral nucleic acids or nucleic acids
`of Viral origin in urine sample, with
`or without
`isolation of nucleic acids
`from a urine sample. The analysis of
`the nucleic acids is performed through
`hybridization of the nucleic acids with
`specific probes, or
`through a chain
`amplification reaction with specific
`primers. The methods are applicable
`to all Viral pathogenic agents, including
`RNA, DNA, episomal, or integrated
`viruses.
`
`
`
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`WO 2006/089203 A2
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`|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes and Abbreviations” appearing at the begin—
`ning of each regular issue of the PCT Gazette.
`
`

`

`WO 2006/089203
`
`PCT/US2006/005792
`
`COMPOSITIONS AND METHODS FOR DETECTING VIRAL SPECIFIC NUCLEIC
`
`ACIDS IN URINE
`
`BACKGROUND OF THE INVENTION
`
`There are currently three types of in vitro diagnostic systems widely used for the detection
`
`of pathogens. These are direct culture of the pathogenic agent fiom the biological sample;
`
`immunological assays based on the detection of products or antigens of the infectious agent; and
`
`indirect immunological assays that can detect antibodies produced against the infectious agent
`
`during infection.
`
`In the first system, the principal disadvantage is that the biological sample must be
`
`considercd to bc at risk for the transmission of the pathogenic agent. In the second and third
`
`systems, the disadvantages include sample retrieval that is often invasive and potentially
`
`infective sample when collected. In the third system, one major disadvantage is that there is
`
`often little possibility of discriminating between past and current infections.
`
`More recently, molecular diagnostic methods have been developed based on the detection
`
`of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell
`
`cultures, taken from the patient. These assays are generally much more sensitive than the
`
`immunological assays. However, they may require the presence of special equipment and
`
`qualified personnel. Furthermore, the biological samples — in the case of plasma, blood, or cell
`
`cultures — are difficult to store unaltered, except under controlled temperature conditions, and
`
`are considered to be biohazardous to personnel who handle them.
`
`Recently, molecular diagnostic methods based on transrenal DNA (Tr—DNA) have been
`
`described for monitoring the progress of allogeneic transplants, to diagnose the sex of a fetus,
`
`and to detect the presence of tumor markers. (Botezatu et al. Clinical Chemistry 46(8):lO78-84
`
`(2000); Su et al. Ann. NY Acad. Sci. 1022:81-89 (2004)) For example, U.S. Patent No.
`
`6,251,638 describes an analytical method for detecting male fetal DNA in the urine of pregnant
`
`women. U.S. Patent No. 6,287,820 describes a system aimed at the diagnosis of tumors,
`
`particularly of adenocarcinomas of the colon and pancreas. U.S. Patent No. 6,492,144 teaches
`
`that the Tr-DNA nucleic-acid analysis method may be used to monitor the progress of allogeneic
`1
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`transplants. The presence of transrenal DNA in urine, in the form of nucleic acid fi‘agments of
`
`fewer than 1000 base pairs was also described in Al—Yatama er al. (2001), Prenat Diagn, 21 :3 99—
`
`402; and Utting, M., et al. (2002), Clin Cancer Res, 8:35-40. Keiko Koide, et al., Prenat Diagn,
`
`2005; 25: 604—607; Mengiun Wang, et al., Clinical Chemistry, 2004, 50: 211-213; Y.-H. Su, et
`
`al., J. M01. Diagn, 2004, 6: 101-107.
`
`10
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`The presence of transrenal DNA has been explained as being the result of phenomenon of
`
`apoptosis. In the process of apoptosis or programmed cell death the nuclear DNA is cleaved into
`
`nucleosomes and oligomers, which subsequently, as a part of apoptotic process, are
`
`phagocytozed and removed from the organism. (Umansky, S.R., et al. (1982); Biochim. Biophys.
`
`Acta, 655:9-17). A portion ofthis degraded DNA, though, escapes the phagocytosis, and appears
`
`in the bloodstream (Lichtenstein, A.V., et al. (2001), Ann NY Acad Sci, 945 1239-249), and, as
`
`confirmed in the above-referred patents, also in urine.
`
`The presence of viral DNA that originates from sources outside of the urinary tract, in
`
`urine had not been described prior to this application. Circulation of Viral DNA released from
`
`the genome of transfected cell in the plasma has been shown. For example, fragments of
`
`Epstein—Barr viral DNA were detected in plasma of patients with nasopharyngeal carcinoma
`
`(Chan, K.C., et‘ al. (2002), Cancer Res 63:2028-2032). Also, human papilloma virus (HPV) viral
`
`DNA has been detected in the plasma of patients with cervical cancer (Pornthanalcasem, W., et
`
`al. (2001); BMC Cancer 1:2). However, these Viral DNAs were not detected in the urine of the
`
`20
`
`patients.
`
`The instant invention describes a method of detecting the presence of viral pathogens in a
`
`subject through the detection of DNA sequences from those pathogens in the urine of the subject.
`
`SUNIIVIARY OF THE INVENTION
`
`25
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`
`The present invention relates to methods for diagnosis and monitoring of viral infections
`
`in a subject by detecting and quantifying transrenal viral nucleic acid—specific sequences in urine
`
`from the subject. In one embodiment, the nucleic acids are isolated or purified. This purification
`
`may be performed using chemical or physical methods. These methods include extraction with
`
`organic solvents, filtration, precipitation, absorption on solid matrices having an affinity for the
`
`nucleic acids, and chromatography. Solid matrices used in these methods may be constructed
`
`from silica—based resins, ion-exchange resins, or hydroxyapatite. In another embodiment, the
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`PCT/US2006/005792
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`solid matrix is a silica—based resin, and said isolation or purification is performed in the presence
`
`of a chaotropic agent. In another embodiment, one or more agents are added to the urine sample
`
`that inhibit the degradation of the nucleic acids. These agents include ion—chelating agents,
`
`denaturing agents, and ionic detergents. An example of an ion—chelating agents used in the
`
`methods of the invention is EDTA. Examples of denaturing agents used in the methods of the
`
`invention are guanidine HCl or guanidine isothiocyanate. Examples of ionic detergents used in
`
`the methods of the invention are N—lauryl sarcosine or sodium dodecyl sulfate. In one
`
`embodiment, isolated or purified nucleic acids are used for the detection of viral nucleic acids.
`
`In other embodiments, the method also includes the fractionation of the urine sample, for
`
`example, through centrifilgation or filtration, with the separation of a cell—free fraction from a
`
`fraction associated with the cell bodies.
`
`The analysis of the nucleic acids is performed using one of the following techniques:
`
`hybridization of the nucleic acids, the cycling probe reaction, a polymerase chain reaction, a
`
`nested polymerase chain reaction (PCR), a semi-nested PCR, single-strand conformation
`
`polymorphism, a ligase chain reaction, strand displacement amplification, and restriction
`
`fragment length polymorphism. In another embodiment, PCR is performed using one or more
`
`primers that are specific for the HIV—1 GAG or POL genes. Examples of these primers are
`
`described in the specification as SEQ ID NOS: 1—1 1.
`
`In another embodiment, the viral nucleic acids are less than about 1000 bp in size. In a
`
`preferred embodiment the Viral nucleic acids are between about 100 and about 200 bp in size.
`
`The methods of the invention are applicable to all viral pathogenic agents, including
`
`RNA, DNA, episomal, and integrative viruses. They also apply to recombinant viruses, such as
`
`the adenoviruses or lentiviruses utilized in gene therapy. In particular, the methods apply to the
`
`following viruses: retroviruses, including recombinant and natural HIV—l, PIN—2, variola virus,
`
`poliovirus, herpes simplex Virus (HSV), Epstein—Barr virus (EBV), hepatitis C virus (HCV),
`
`hepatitis B virus (I-IBV) and adenoviruses (AAV). In some embodiments the Viral agents are
`
`Epstein—Barr virus (EBV) and HIV—1. In one embodiment, the methods of the invention are
`
`performed in vitro.
`
`In another embodiment, the invention relates to a method for monitoring a viral infection
`
`in a subject, including the steps of quantitating the amount of a transrenal viral nucleic acid in a
`
`first urine sample from said subject; quantitating the amount of said transrenal viral nucleic acid
`
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`in a second urine sample from said subject; and comparing the amount of said transrenal viral
`
`nucleic acid in said first and said second urine sample from said subject, thereby monitoring said
`
`Viral infection in said subject. In another aspect of this embodiment, the method further
`
`comprises the step of administering to said subject a compound that reduces or inhibits said viral
`
`infection. Optionally, the viral infection is HIV infection and said compound is an anti-viral
`
`agent. In another aspect of this embodiment, the quantitating is performed by a means selected
`
`from pairing with molecular probes that are specific for those pathogenic agents, hybridization,
`
`PCR, nested PCR, semi-nested PCR, SSCP, LCR, and SDA. In another aspect of this
`
`embodiment, the method further comprises the step of separating said urine samples into a cell—
`
`free fraction containing said transrenal nucleic acids. This separation may be done using
`
`centrifugation. The nucleic acids of this embodiment may be between 100 and 200 bp. In
`
`another aspect of this embodiment, the subject is at risk for developing a recurring viral infection.
`
`In another of its embodiments, the invention relates to a kit for the detection of viral
`
`nucleic acid in urine, including: reagents and/or materials for the fractionation and/or extraction
`
`of transrenal viral nucleic acids from urine, DNA probe, or pairs of specific oligonucleotides
`
`(primers) for at least one Viral agent. In one aspect of this embodiment, the probe is a primer for
`
`a PCR reaction that is specific for the HIV-1 GAG or POL genes. Examples ofthese primers are
`
`described in the specification as SEQ ID NOS: 1-1 1.
`
`Unless otherwise defined, all technical and scientific terms used herein have the same
`
`meaning as commonly understood by one of ordinary skill in the art to which this invention
`
`relates. Although methods and materials similar or equivalent to those described herein can be
`
`used in the practice or testing ofthe present invention, suitable methods and materials are
`
`described below. All publications, patent applications, patents, and other references mentioned
`
`herein are incorporated by reference in their entirety. In the case of conflict, the present
`
`specification, including definitions, will control. In addition, the materials, methods, and
`
`examples are illustrative only and not intended to be limiting.
`
`Other features and advantages of the invention will be apparent from the following
`
`detailed description and claims.
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`BRIEF DESCRIPTION OF THE FIGURES
`
`Figure 1 is a photographic image showing the results of gel electrophoresis of the
`
`amplification product obtained through nested PCR of the transrenal DNA of non-fractionated
`
`urine samples of patients infected with HIV—1 (20 cycles with the 468/602 primer pair [134 bp]
`
`followed by 35 cycles with 518/596 [79 bp]). The primers are specific for the HIV—1 GAG
`
`region.
`
`Figure 2 is a photographic image showing the results of gel electrophoresis of the
`
`amplification product obtained through nested PCR of the genomic DNA extracted from 8E5
`
`LAV cells (20 cycles with the 468/602 primer pair [134 bp] followed by 35 cycles with 518/596
`
`[79 bp]). The primers are specific for the HIV-1 GAG region.
`
`Figure 3 is a photographic image showing the results of gel electrophoresis of the
`
`amplification product obtained through nested PCR of the transrenal DNA of urine samples of
`
`patients infected with HIV—1 (CP: complete; SN: supernatant; PT: pellet). A—102bp/60bp
`
`(specific primers for the HIV—1 POL region); B-317bp/60bp (specific for POL); C-569bp/132bp
`
`(specific for TAT); NI: non-infected; IN 1-3: patients infected with HIV.
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`DETAILED DESCRIPTION OF THE INVENTION
`
`The present definitions are offered for the purposes of the present invention:
`
`Amplicon: A term for any relatively small, DNA fragment that is replicated, e. g., by
`
`20
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`PCR.
`
`Amplification: An increase in the number of copies of a specific DNA fragment can
`
`occur in vivo or in vitro.
`
`Apoptosis: Programmed cell death in normally functioning human and animal cells when
`
`age or state of cell health and condition dictate. An active process requiring metabolic activity by
`
`25
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`the dying cell, characterized by cleavage of the DNA into fragments that give a so called
`
`laddering pattern of DNA fiagments of nucleosomal size and its oligomers.
`
`Chaotropic: The property of chemical substances (e.g., ions such as SCN, ClO4‘, and
`
`guanidine) that disturb the thermodynamic structure of water. It allows less polar and more
`
`hydrophobic substances to become more soluble in water. The effect at the biological level is the
`
`30
`
`denaturation of proteins.
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`WO 2006/089203
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`Episome: A circular DNA molecule of that can replicate independently of the cellular
`
`chromosome or integrate and replicate as part of the chromosome.
`
`Gene: DNA fragment that contains sequences necessary to code for an mRNA, and to
`
`control the expression of these sequences.
`
`Genome: The total set of genes of an organism enclosed, among the eukaryotes, in
`
`chromosomal structures.
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`Cyclic Probe Reaction: CPT reactions are performed at a constant specific temperature,
`
`which allows hybridization of the chimeric probe with its complementary single—stranded target
`
`DNA. Within the resulting target—probe duplex, RNase H recognizes the DNA—RNA hybrid and
`
`specifically cleaves the RNA portion of the probe. The cleaved fragments are not stable at the
`
`reaction temperature and disassociate from the target. The target is then free to hybridize with
`
`another probe molecule, and the cycle is repeated. The probe fragments accumulate, serving as a
`
`basis for the detection of target. Over time, the accumulation of cleaved probe fragments follows
`
`linear kinetics and therefore the amount of target can be quantified.
`
`Hybridization: A widely used technique that exploits the ability of complementary
`
`sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix.
`
`Hybridization can take place between two complimentary DNA sequences, between a single-
`
`stranded DNA and a complementary RNA, or between two RNA sequences. The technique is
`
`used to detect and isolate specific sequences, measure homology, or define other characteristics
`
`20
`
`of one or both strands.
`
`Infection: Invasion and multiplication of microorganisms in body tissues, which may be
`
`clinically unapparent or result in local cellular injury due to competitive metabolism, toxins,
`
`intracellular replication or antigen antibody response.
`
`Ligase Chain Reaction: A method of DNA amplification similar to PCR. LCR differs
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`25
`
`from PCR because it amplifies the probe molecule rather than producing amplicon through
`
`polymerization of nucleotides. Two probes are used per each DNA strand and are ligated together
`
`to form a single probe. LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to
`
`drive the reaction. Like PCR, LCR requires a thermal cycler to drive the reaction and each cycle
`
`results in a doubling of the target nucleic acid molecule. LCR can have greater specificity than
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`Nested PCR: A second PCR that is performed on the product of an earlier PCR using
`
`primer, which are internal to the originals. This significantly improves the sensitivity and
`
`specificity of the PCR.
`
`Nested primer: A selected primer internal to an amplicon obtained with a first PCR
`
`cycle. The amplification process that uses at least one nested primer improves specificity,
`
`because the non-specific products of the first cycle are not amplified in the second cycle.
`
`Nucleic Acid: Linear polymers of nucleotides, linked by 3', 5' phosphodiester linkages. In
`
`DNA, deoxyribonucleic acid, the sugar group is deoxyribose and the bases of the nucleotides
`
`adenine, guanine, thymine and cytosine. RNA, ribonucleic acid, has ribose as the sugar and uracil
`
`replaces thymine. DNA functions as a stable repository of genetic information in the form of base
`
`sequence. RNA has a similar function in some viruses but more usually serves as an
`
`informational intermediate (mRNA), a transporter of amino acids (tRNA), in a structural capacity
`
`or, in some newly discovered instances, as an enzyme.
`
`Oligonucleotide/Polynucleotide: Linear sequence of two or more nucleotides joined by
`
`phosphodiester bonds. Above a length of about 20 nucleotides the term “polynucleotide” is
`
`generally used.
`»
`Pathogenic agent: A pathogen is a biological agent that can cause disease to its host. A
`
`synonym of pathogen is "infectious agent". The term "pathogen" is most often used for agents
`
`that disrupt the normal physiology of a multicellular organism.
`
`Pellet: Sediment, when cells are present, usually includes the cell fraction, or that can be
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`obtained by centrifuging a biological sample.
`
`Polymerase: Enzyme utilized in the amplification of nucleic acids. The term includes all
`
`of the variants of DNA polymerases.
`
`Primer: Short pre-existing polynucleotide chain to which new deoxyribonucleotides can
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`be added by DNA polymerase.
`
`PCR: Polymerase Chain Reaction involving two synthetic oligonucleotide primers, which
`
`are complementary to two regions of the target DNA (one for each strand) to be amplified, are
`
`added to the target DNA (that need not be pure), in the presence of excess deoxynucleotides and
`
`Taq polymerase, a heat stable DNA polymerase. In a series (typically 30) of temperature cycles,
`
`30
`
`the target DNA is repeatedly denatured (around 90 °C), annealed to the primers (typically at 50-
`
`60 °C) and a daughter strand extended ficm the primers (72°C). As the daughter strands
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`themselves act as templates for subsequent cycles, DNA fragments matching both primers are
`
`amplified exponentially, rather than linearly.
`
`Probe: General term for a fiagment of DNA or RNA corresponding to a gene or sequence
`
`of interest that has been labelled either radioactively or with some other detectable molecule,
`
`such as biotin, digoxygenin or fluorescein.
`
`Purification / Decontamination / Sterilization: Refers to a process for removing
`
`contaminants from a sample, where the result is a sample containing 60%, preferably 75%, and
`
`even more preferably 90% of the material toward which the purification procedure is directed.
`
`Restriction Fragment Length Polymorphism (RFLP): A method that allows genetic
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`relationship established by comparing the characteristic polymorphic patterns that are obtained
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`when certain regions of genomic DNA are amplified (typically by PCR) and cut with certain
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`restriction enzymes. Variations in such patterns are generated by mutations that create or abolish
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`recognition sites for these enzymes
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`Sample: The term is broadly interpreted and includes any form that contains nucleic acids
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`(DNA or RNA) in solution or attached to a solid substrate, where the definition of “nucleic
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`acids” includes genomic DNA (for example, when it is attached to a solid substrate, such as in
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`the Southern Blot or in solution), cDNA, and other forms.
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`Combinations of two nucleic-acid sequences through hybridization are formed thanks to
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`the hydrogen bonds between G and C or A and T bases or analogs of these bases. These
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`combinations are complementary, and the DNA helixes are anti-parallel. This hybridization
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`combination can be created with one sequence (or helix) in a solution and the other attached to a
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`solid phase (such as, for example, in the FISH [fluorescent in situ hybridization] method), or else
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`with both of the sequences in solution.
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`Single-Strand Conformation Polymorphism (SSCP): SSCP is the electrophoretic
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`separation of single-stranded nucleic acids based on subtle differences in sequence (often a single
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`base pair) that results in a different secondary structure and a measurable difference in mobility
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`through a gel.
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`Strand Displacement Amplification (STA): STA is an isothermal, in vitro nucleic acid
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`amplification technique based upon the ability of HincII to nick the unmodified strand of a
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`hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient
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`Klenow fragment of DNA Polymerase (exo— klenow) to extend the 3'-end at the nick and displace
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`the downstream DNA strand. Exponential amplification results from coupling sense and
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`antisense reactions in which strands displaced from a sense reaction serve as target for an
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`antisense reaction and Vice versa.
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`Target sequence: Nucleic-acid sequence that should be analyzed through hybridization,
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`amplification, or other methods or combinations of methods.
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`Tm (melting temperature): Temperature at which a specific double-helix DNA
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`population dissociates into single-strand polymers. The formula for calculating this temperature
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`for polynucleotide fragments is well known in the art: Tm = 81.5 + 0.41 (% G+C) (Anderson &
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`Young, “Quantitative Filter Hybridization,” in Nucleic Acid Hybridization [1985]). For
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`oligonucleotides with fewer than 40 base pairs, a simplified formula can be used: Tm = 3°C x (G
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`+C)+2x(A+T).
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`Tr—DNA/RNA: Transrenal DNA/RNA, or DNA/RNA present in urine afler having been
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`passed through the kidney barrier.
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`Urinary tract: Includes the organs and ducts that participate in the elimination of urine
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`from the body.
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`Urinary nucleic acids in viral pathogen infections
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`The present invention is based on the discovery that following a Viral infection, the
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`nucleic acids of the Virus(es) or of Viral origin are cleaved into relatively short fragments which
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`are found in the urine. Many of these pathogen specific nucleic acids cross the transrenal barrier
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`(these nucleic acids are generally termed TrNA, or TrDNA or TrRNA) and can be detected in
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`urine as cell-free low-molecular-weight fragments (whose length is less than 1000 nucleotides,
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`but are preferably less than 500 bp in length, and more preferably shorter than 250—300 bp in
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`length or shorter than 250 bp in length) through molecular methods. These transrenal nucleic
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`acids are derived from viruses which are located outside of the urinary tract of a subject. As used
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`herein, the term “Viral nucleic acid” encompasses nucleic acids of Viral origin. Other virus
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`specific nucleic acids may be shed by Virus or cells that are within the kidney, and thus do not
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`have to cross the transrenal barrier in order to be detected in the urine. Further, some virus
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`specific nucleic acids may be found in the urine through other mechanisms besides crossing the
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`transrenal barrier or being generated by Virus in the kidney.
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`The presence of transrenal nucleic acids (Tr—NA) in urine was detected previously only in
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`hosts who had undergone heterologous tissue or organ transplants, in the case of women pregnant
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`with male fetuses, and in the case oftumors characterized by specific marker genes. The
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`presence of transrenal nucleic acids of viral origin in the case of viral infections according to the
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`present invention is also, and preferably, detected in the case ofnon— urinary-tract infections,
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`even in the absence of hematuria or of pathologies that lead to the rupture, or that alter the
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`normal integrity, of the renal barrier.
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`Transrenal nucleic acids (Tr-NA) of viral origin are not associated with, and are not
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`derived from, the genome of viruses that are lost or released in the urinary tract and that are
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`found in urine. Instead, transrenal nucleic acids are filtered by the glomerular—renal filtration
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`mechanism. Thus, the dimensions of the transrenal nucleic—acid fragments are generally smaller
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`than about 1000 base pairs, e. g., smaller than about 500, smaller than about 300, smaller than
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`about 250, or between about 100 and about 200 base pairs, as opposed to other situations in
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`which DNA usually has a high molecular weight and a length in excess of 1000 bases or base
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`pairs.
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`Therefore, in the present invention, the transrenal nucleic acid (TrNA) of viral origin is
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`generally not found in the urine sediment, but in the soluble fraction, although traces of TrNA
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`can co-sediment with the cells during centrifuging.
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`The discovery confirms the presence of urinary nucleic acids or transrenal nucleic acids
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`derived from pathogenic viruses in urine, and therefore is applicable to the diagnosis of all
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`infectious diseases caused by viral pathogens.
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`Therefore, in embodiments, the invention relates to methods for diagnosis or monitoring
`of viral infection by determining the presence of viral nucleic acids, preferably viral DNA or
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`RNA of viral origin, in a urine sample. The methods includes the step of determining the
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`presence oftransrenal Viral nucleic acids using methods generally used in laboratory practice
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`such as hybridization, PCR, nested PCR, semi-nested PCR, SSCP, LCR, and SDA.
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`In certain embodiments, the methods according to the invention include an initial
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`treatment of the urine sample prior to the determination of the presence of transrenal viral nucleic
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`acids. In an embodiment, the invention includes the pretreatment ofthe urine sample with an
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`agent that inhibits the degradation ofthe DNA or RNA. These agents include the enzymatic
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`inhibitors, such as chelating agents, detergents, or denaturing agents, DNase or RNase inhibitors,
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`which are preferably selected from the group consisting of EDTA, guanidine HCl, guanidine
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`isothiocyanate, N—lauryl sarcosine, and sodium dodecyl sulfate.
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`In another embodiment, the determination of the presence of transrenal viral nucleic acids
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`optionally is preceded by centrifugation or filtration of the urine sample in order to separate the
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`cellular fraction of the urine from the cell-free low-molecular—weight nucleic acids (DNA/RNA).
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`However, the urine sample may also be utilized without fractionation. Centrifugation is
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`preferably performed at a speed between 25 00g and 45 00g, and more preferably between 3000g
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`and 4000g. Filtration is preferred to carry out through a filter with pore size between 0.1 and 5.0
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`um, more preferably with pore size between 0.2 and 1.0 um and even more preferably 0.45 and
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`0.8 um. Equivalent methods for separating the soluble fraction from the cellular fraction may
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`also be used.
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`The optional isolation or purification and quantification of the transrenal nucleic acids are
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`achieved through the use of chemical or physical methods that are already known in the art. It
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`includes at least one purification step, using methods selected from among extraction with
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`organic solvents, filtration, precipitation, absorption on solid matrices (e.g., Via ion exchange),
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`affinity chromatography or else molecular exclusion chromatography or combinations of these
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`methods.
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`However, the purification method must be appropriate for the isolation of DNA (single-
`
`or double-helix) that are less than 1000 nucleotides in length, with a corresponding molecular
`
`weight, assuming, as the average molecular weight, that of a nucleotide having a value of 330
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`Daltons. In some embodiments, the purification is specific for fragments that are smaller than
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`500 nucleotides (nt) in length, with a corresponding molecular weight, such as fiagments whose
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`lengths are less than 300 nt, fiagments less than 250 nt in length, or fragments whose lengths are
`
`between 100 and 200 base pairs of nucleic acids (nt).
`
`The isolation and/or purification of the transrenal nucleic acids is achieved through the
`
`use of chemical or physical methods that are already known in the art. It includes one or more
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`purification steps using methods selected fi‘om among extraction with organic solvents, filtration,
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`precipitation, absorption on solid matrices (e.g., silica resin, hydroxyapatite or ion exchange),
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`affinity chromatography